The present invention relates to the field of immunology and is particularly concerned with adjuvants, i.e. materials which modulate immune response to an antigen.
Vaccines have been used for many years to protect humans and animals against a wide variety of infectious diseases. Such conventional vaccines consist of attenuated pathogens (for example, polio virus), killed pathogens (for example, Bordetella pertussis) or immunogenic components of the pathogen (for example, diphtheria toxoid). Some antigens are highly immunogenic and are capable alone of eliciting protective immune responses. Other antigens, however, fail to induce a protective immune response or induce only a weak immune response.
In the development of some vaccines and immunogenic compositions, there is a trend to use smaller and well defined immunogenic and protective materials. Recent advances in molecular genetics, protein biochemistry, peptide chemistry, and immunobiology have provided economical and efficient technologies to identify and produce large quantities of pure antigens from various pathogens. However, some such materials may not be sufficiently immunogenic, due to either their small size (especially synthetic peptides) or the lack of intrinsic immunostimulatory properties thereof.
Immunogenicity can be significantly improved if the antigens are co-administered with adjuvants. Adjuvants enhance the immunogenicity of an antigen but are not necessarily immunogenic themselves. Adjuvants may act by retaining the antigen locally near the site of administration to produce a depot effect facilitating a slow, sustained release of antigen to cells of the immune system. Adjuvants can also attract cells of the immune system to an antigen depot and stimulate such cells to elicit immune responses.
Immunostimulatory agents or adjuvants have been used for many years to improve the host immune responses to, for example, vaccines. Intrinsic adjuvants, such as lipopolysaccharides, normally are the components of the killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Thus, adjuvants have been identified that enhance the immune response to antigens delivered parenterally. Some of these adjuvants are toxic, however, and can cause undesirable side-effects, making them unsuitable for use in humans and many animals. Indeed, only aluminum hydroxide and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants in human and veterinary vaccines. The efficacy of alum in increasing antibody responses to diptheria and tetanus toxoids is well established and, more recently, a HBsAg vaccine has been adjuvanted with alum. While the usefulness of alum is well established for some applications, it has limitations. For example, alum is ineffective for influenza vaccination and inconsistently elicits a cell mediated immune response. The antibodies elicited by alum-adjuvanted antigens are mainly of the IgG1 isotype in the mouse, which may not be optimal for protection by some vaccinal agents.
A wide range of extrinsic adjuvants can provoke potent immune responses to antigens. These include saponins complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria in mineral oil, Freund""s complete adjuvant, bacterial products, such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.
To efficiently induce humoral immune responses (HIR) and cell-mediated immunity (CMI), immunogens are emulsified in adjuvants. Many adjuvants are toxic, inducing granulomas, acute and chronic inflammations (Freund""s complete adjuvant, FCA), cytolysis (saponins and Pluronic polymers) and pyrogenicity, arthritis and anterior uveitis (LPS and MDP). Although FCA is an excellent adjuvant and widely used in research, it is not licensed for use in human or veterinary vaccines because of its toxicity.
Desirable characteristics of ideal adjuvants include:
(1) lack of toxicity;
(2) ability to stimulate a long-lasting immune response;
(3) simplicity of manufacture and stability in long-term storage;
(4) ability to elicit both CMI and HIR to antigens administered by various routes, if required;
(5) synergy with other adjuvants;
(6) capability of selectively interacting with populations of antigen presenting cells (APC);
(7) ability to specifically elicit appropriate TH1 or TH2 cell-specific immune responses; and
(8) ability to selectively increase appropriate antibody isotype levels (for example, IgA) against antigens.
U.S. Pat. No. 4,855,283 granted to Lockhoff et al on Aug. 8, 1989 teaches glycolipid analogues including N-glycosylamides, N-glycosylureas and N-glycosylcarbamates, each of which is substituted in the sugar residue by an amino acid, as immuno-modulators or adjuvants. Thus, Lockhoff et al. (U.S. Pat. No. 4,855,283) reported that N-glycolipid analogs displaying structural similarities to the naturally-occurring glycolipids, such as glycosphingolipids and glycoglycerolipids, are capable of eliciting strong immune responses in both herpes simplex virus vaccine and pseudorabies virus vaccine. Some glycolipids have been synthesized from long chain-alkylamines and fatty acids that are linked directly with the sugars through the anomeric carbon atom, to mimic the functions of the naturally occurring lipid residues.
U.S. Pat. No. 4,258,029 granted to Moloney, assigned to the assignee hereof, teaches that octadecyl tyrosine hydrochloride (OTH) functioned as an adjuvant when complexed with tetanus toxoid and formalin inactivated type I, II and III poliomyelitis virus vaccine. Also, Nixon-George et al. (1990), J. Immunology 144:4798-4802 reported that octadecyl esters of aromatic amino acids complexed with a recombinant hepatitis B surface antigen, enhanced the host immune responses against hepatitis B virus.
Lipidation of synthetic peptides has also been used to increase their immunogenicity. Thus, Wiesmuller ((1989), Vaccine 7:29-33) describes a peptide with a sequence homologous to a foot-and-mouth disease viral protein coupled to an adjuvant tripalmityl-S-glyceryl-cysteinylserylserine, being a synthetic analogue of the N-terminal part of the lipoprotein from Gram negative bacteria. Furthermore, Deres et al. (1989, Nature 342:561) reported in vivo priming of virus-specific cytotoxic T lymphocytes with synthetic lipopeptide vaccine which comprised of modified synthetic peptides derived from influenza virus nucleoprotein by linkage to a lipopeptide, N-palmityl-S-[2,3-bis(palmitylxy)-(2RS)-propyl-[R]-cysteine (TPC).
The adjuvants and immunostimulating compounds described above may not provide for adjuvanticity for all antigens delivered to a variety of hosts under many conditions.
It would be desirable to provide adjuvant compositions that do not suffer from the disadvantages and limitations of currently available adjuvants.
The present invention is directed towards the provision of improved adjuvant compositions. In accordance with one aspect of the present invention, there is provided an adjuvant composition for modulating an immune response to an antigen administered to a host, the composition comprising:
(a) a mineral salt adjuvant; and
(b) at least one other adjuvant.
The multiple adjuvant compositions provided herein exhibit a surprisingly unexpected adjuvanting effect on an antigen which is greater than the adjuvanting effect attainable by one of the adjuvants alone. The enhanced effect may be additive of the adjuvanting effect of the individual adjuvants and, in particular embodiments, a synergistic effect is attained.
The mineral salt adjuvant preferably comprises aluminum hydroxide or aluminum phosphate, although other known mineral salt adjuvants, such as calcium phosphate, zinc hydroxide or calcium hydroxide, may be used. The at least one other adjuvant may be a glycolipid analog, an octadecyl ester of an amino acid (such as an aromatic amino acid) or a lipoprotein. The lipoprotein may be a synthetic analogue of an N-terminal portion of lipoprotein.
In a particular embodiment, the glycolipid may be a glycosylamide and may have the formula: 
wherein R1 denotes hydrogen or saturated or singly or multiply unsaturated alkyl radical having up to 50 carbon atoms;
X represents xe2x80x94CH2xe2x80x94, xe2x80x94Oxe2x80x94 or xe2x80x94NHxe2x80x94;
R2 denotes hydrogen or a saturated or singly or multiply unsaturated alkyl radical having up to 50 carbon atoms, R3, R4, and R5, independently of one another, denotes hydrogen, or SO42xe2x88x92, or PO42xe2x88x92, or other negatively charged moiety, or xe2x80x94COxe2x80x94R6, R6 being an alkyl radical having up to 10 carbon atoms;
R7 is L-alanyl, L-alpha-aminobutyryl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxypropyl, L-isoleucyl, L-leucyl, L-lysyl, L-methionyl, L-ornithinyl, L-phenylalanyl, L-prolyl, L,-seryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl or their D-isomers;
and pharmaceutically acceptable salts thereof. In an embodiment, the glycosylamide may be N-(2-deoxy-2-L-leucylamino-xcex2-D-glucopyranosyl)-N-octadecyldodecanamide acetate.
In accordance with a further aspect of the present invention, there is provided an immunogenic composition for eliciting an immune response in a host, including a human, the composition comprising:
(a) at least one antigen;
(b) a mineral salt adjuvant; and
(c) at least one other adjuvant.
In yet another aspect of the invention, there is provided a method of modulating an immune response to an antigen comprising combining the antigen with the adjuvant composition of the present invention.
Convenient antigens which may be included in said immunogenic compositions and in respect of which an immune response is modulated, include microbial pathogens, bacteria, viruses, proteins, glycoproteins lipoproteins, peptides, glycopeptides, lipopeptides, toxoids, carbohydrates, and tumor-specific antigens. Mixtures of two or more antigens may be employed.
Such peptides, glycopeptides or lipopeptides may include an amino acid sequence corresponding to an antigenic determinant of HIV, Rubella virus, Respiratory Syncytial Virus, Bordetella pertussis, Haemophilus influenzae or Streptocococcus pneumoniae, including those specific synthetic peptides shown in Table I below (SEQ ID NOS: 1 to 15) (The Tables appear at end of the descriptive text) or a functional analog thereof. The toxoid may be a pertussis toxoid while the protein may be influenza hemagglutinin or a parainfluenza virus subunit, such as the HN or F proteins of PIV-3.
In a further aspect of the invention, there is provided a method of generating an immune response in a host, including a human, comprising administering thereto the immunogenic composition of the present invention. The immune response attained may be a humoral or a cell-mediated immune response.
In a particular aspect of the present invention, there is provided a kit for preparing an immunogenic composition, comprising:
(a) means for containing a mineral salt adjuvant;
(b) means for containing at least one other adjuvant;
(c) means for containing at least one antigen; and
(d) means for combining the mineral salt adjuvant, at least one other adjuvant and at least one antigen to produce the immunogenic composition.
In a further aspect of the present invention, there is provided a compound comprising an antigen, including any of those referred to above, covalently linked to a glycolipid analog, such as a glycosylamide, as well as immunogenic compositions comprising the same for generating an immune response in a host, including a human. The glycosylamide may have the formula I above.
By covalently bonding an antigen to a glycolipid analog, a discrete molecule is produced which exhibits a surprisingly unexpected enhanced adjuvanting effect on the antigen which is greater than the adjuvanting effect attainable in the absence of such covalent bonding, as in a mixture of the two components. A further enhanced adjuvanting effect may be attained for such covalently-bonded antigen by incorporating a mineral salt adjuvant with such compounds.
The antigen may be covalently linked to the glycolipid analog at a carboxy or amino terminus or other suitable site compatible with covalent linkage of the antigen by, for example, a cross-linker having a reactive function, such as maleimidyl, succinimidyl, 2-pyridyldithio, NH2, SH, and xe2x80x94COxe2x80x94R8, where R8 is xe2x80x94OH, N3, xe2x80x94O-alkyl(C1-C2), xe2x80x94OC6F5, H, Br, or Cl.
Advantages of the present invention include:
(a) ease of formulation;
(b) effectiveness of adjuvanticity; and
(c) compatibility of antigens with the adjuvant composition.